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Author ORCID Identifier

https://orcid.org/0000-0001-9725-9286

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

September

First Advisor

Mingxu You

Subject Categories

Analytical Chemistry | Biochemistry, Biophysics, and Structural Biology

Abstract

Lipid-DNA conjugates are becoming popular for cell membrane engineering, regulation, and analysis. Recent progress in bioconjugate chemistry and synthetic biology has further promoted the fast growing of this field. Taking advantage of these lipide-DNA conjugates, different functional moieties can be efficiently engineered onto live cell membranes, with minimum effect on natural cellular behavior. These conjugates exhibit several attractive features such as fast and efficient membrane insertion after simple incubation, universality toward different cell types, precise hybridization and dynamic switching of oligonucleotides and versatile functionality. Even though Lipid-DNA conjugates have demonstrated great potential for membrane applications, there are several questions and challenges including our limited understanding on the effect of lipid and DNA structure in their membrane insertion and stability, their influence on the cell membrane function and their limited membrane persistence.

Here, we first aim to answer some of these questions and further improve the lipid-DNA conjugates functions on the cell membrane. Our results indicated that the hydrophobicity of the lipid–DNA probes is strongly correlated with their membrane insertion and departure rates. Most cell membrane insertion stems from the monomeric form of probes, rather than the aggregates. Lipid-DNA probes can be removed from cell membranes through either endocytosis or direct outflow into the solution. As a result, long-term probe modifications on cell membranes can be realized in the presence of excess probes in the solution and/or endocytosis inhibitors. For the first time, we have successfully improved the membrane persistence of lipid-DNA probes to more than 24 h. Our quantitative data have dramatically improved our understanding of how lipid-DNA probes dynamically interact with cell membranes. These results can be further used to allow a broad range of applications of lipid-DNA probes for cell membrane analysis and regulation.

Next, we introduced a DNA-based probe, termed “DNA Zipper”, which allows the membrane pattern of transient lipid–lipid interactions to be imaged in living cells using standard fluorescence microscopes. The DNA Zipper can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. By fine-tuning the length and binding affinity of DNA duplex, the DNA Zipper probe allows us to image membrane cholesterol–cholesterol interactions and lipid domains. The correlation between membrane lipid domain formation and the activation of T-cell receptor signaling has also been studied. Since the DNA Zipper is functional after a brief cell incubation, we believe these programmable and commercially available DNA probes can be easily adopted to study lipid interactions and domain formations during different membrane signaling events.

DOI

https://doi.org/10.7275/24274894

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Thursday, September 01, 2022

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